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Bignoniaceae) N. M. Apóstolo et al., In vitro an exBONPLA vitro germinationNDIA 25(1): of Handroanthus 5-15. 2016 ISSN: 0524-0476 IN VITRO AND EX VITRO GERMINATION OF THREE HANDROANTHUS SPECIES (BIGNONIACEAE) NANCY M. APÓSTOLO1, EZEQUIEL E. LARRABURU1, MIRIAM N. GIL2, MARÍA A. ZAPATER2 & BERTA E. LLORENTE1 Summary: Apóstolo, N. M., E. E. Larraburu, M. N. Gil, M. A. Zapater & B. E. Llorente. 2016. In vitro and ex vitro germination of three Handroanthus species (Bignoniaceae). Bonplandia 25(1): 5-15. Handroanthus impetiginosus, H. ochraceus and H. lapacho, “lapachos”, are found in the Northwest of Argentina and have germination and conservation problems in their natural habitats. The germination of seeds under controlled conditions is an alternative that ensures the propagation. In this integrated study, in vitro and ex vitro germination, seed characteristics and seedling morphology of three species were analyzed. The length and width of the seeds, the width of the wings, the width and length of the seed body and embryo were measured. The germination capacity of the three species was determined during 12 months after seed harvesting, carrying out ex vitro and in vitro germination trials. The seeds and embryos of H. impetiginosus were significantly larger than those of other species. Handroanthus impetiginosus seeds maintained a high germination capacity over the 12 months (95%). Seed germination capacity of H. ochraceus gradually decreased four months after seed collection (63 to 47%), whereas that of H. lapacho decreased abruptly after eight months. No significant differences were observed between ex vitro and in vitro methods in H. impetiginosus and H. ochraceus. In the three species, the germination capacity is directly proportional to the size of seeds and seedlings. Key words: Handroanthus impetiginosus, H. lapacho, H. ochraceus, seed, seedling. Resumen: Apóstolo, N. M., E. E. Larraburu, M. N. Gil, M. A. Zapater & B. E. Llorente. 2016. Germinación in vitro and ex vitro de tres especies de Handroanthus (Bignoniaceae). Bonplandia 25(1): 5-15. Handroanthus impetiginosus, H.ochraceus and H. lapacho, "lapachos”, se distribuyen en el NO Argentino y presentan inconvenientes de germinación y conservación en su ambiente natural. La germinación de semillas bajo condiciones controladas es una alternativa para asegurar la propagación de especies con este tipo de problemáticas. En el presente estudio integral, se analizó la germinación in vitro y ex vitro, las características de las semillas y la morfología de las plántulas de las tres especies de Handroanthus mencionadas. Para ello, se midió el largo y ancho de las semillas, el ancho de las alas de la cubierta seminal, el ancho y largo del cuerpo seminal y del embrión. El poder germinativo de las tres especies fue determinado durante 12 meses luego de la cosecha de las semillas. Fueron determinados los parámetros de las plántulas obtenidas in vitro y ex vitro. El tamaño de la semilla y embrión de H. impetiginosus 1 Laboratorio de Cultivo de Tejidos Vegetales (CULTEV), Dep. de Ciencias Básicas, Universidad Nacional de Luján, C.C. 221. Luján (6700), Buenos Aires, Argentina. E-mail: [email protected] 2 Facultad de Ciencias Naturales, Universidad Nacional de Salta, Av. Bolivia 5150, Salta (4400), Argentina. 5 BONPLANDIA 25(1). 2016 fue significativamente mayor que el de las otras especies estudiadas.H. impetiginosus mantuvo altos porcentajes de germinación hasta los 12 meses (95 %). H. ochraceus perdió gradualmente su poder germinativo a partir de los 4 meses posteriores a la cosecha de las semillas (63 a 47 %). Mientras que, en H. lapacho disminuyó abruptamente a los 8 meses luego de la cosecha. No se observaron diferencias significativas entre ambos tratamientos de germinación (in vitro y ex vitro) para H. impetiginosus y H. ochraceus. Las tres especies mostraron una relación directa entre la capacidad germinativa y las dimensiones de las semillas y características de las plántulas. Palabras clave: Handroanthus impetiginosus, H. lapacho, H. ochraceus, plántula, semilla. Introduction Handroanthus lapacho is a tree up to 20 m in height, with yellow flowers, and The genus Handroanthus belongs to the its distribution is restricted to a narrow Bignoniaceae family and arose from the altitudinal belt (1400-1750 m) in the north segregation of the genus Tabebuia Gomes of Argentina (Jujuy and Salta). Even though ex DC. by molecular phylogenetic studies it is naturally protected, it is considered as (Grose & Olmstead, 2007a, b). Currently, the vulnerable by the International Union for species included in Tabebuia (Gentry, 1972) Conservation of Nature (IUCN) as it occurs are distributed in three genera: Roseodendron in a geographically fragmented area and Miranda, Handroanthus Mattos and Tabebuia its occurrence and quality of habitat are Gomes ex DC. In Argentina, there are only two declining. It is cultivated sporadically in species in Tabebuia: T. aurea and T. nodosa, and urban areas in Salta and Jujuy (IUCN, 2004; none in Roseodendron. Most of the native species Zapater et al., 2009). belong to Handroanthus: H. impetiginosus The seeds of Handroanthus are thin, (Mart. ex DC) Mattos, H. lapacho (K. Schum.) exalbuminous and have a winged seed S. Grose and H. ochraceus (Cham.) Mattos are coat. The seed body is cordiform, flat and distributed in northwest Argentina, whereas H. wide (Silva et al., 2004; Ferreira & Cunha, albus (Cham.) Mattos, H. pulcherrimus (Sandw.) 2000; Zapater et al., 2009). Costa et al. S. Grose and H. heptaphyllus (Vell.) Mattos are (2004) and Sampaio et al. (2007) have found in the northeast (Grose & Olmstead, studied the ontogeny of H. ochraceus seed. 2007b; Lozano & Zapater, 2008; Zapater et al., Morphological and physiological aspects 2009; Zuloaga et al., 2011). of the germination of H. impetiginosus Handroanthus impetiginosus is a tree 20-30 have been reported by Silva et al. (2004). m in height, with pink flowers, and it is found Morphological studies of the fruit, seed and up to 1,300 m in the Tucumano-Oranense seedlings have been reported by different forest in the provinces of Jujuy, Salta, Tucumán authors (Corner, 1976; Carvahlo et al., 2008; and Catamarca, Argentina. It has the widest Ferreira & Cunha 2000; Souza et al., 2005). distribution in Argentina of the species in this Even though most species of Handroanthus genus and it is cultivated as an ornamental produce large quantities of seed, many of from northwest Argentina to the province of them have germination and conservation Buenos Aires. However, its natural area has problems in their natural habitats (Bocchese been reduced by anthropogenic activities (Silva et al., 2008; Carvahlo et al., 2008; Justiniano et al., 2004; Lozano & Zapater, 2008; Zapater et et al., 2000; Nery et al., 2008). In vitro al., 2009). germination has been little studied (Nery Handroanthus ochraceus is a tree of 30-35 et al., 2008), however some studies have m in height, with yellow flowers, and its range been carried out on the ex situ germination of distribution covers Salta and Jujuy provinces of several species of this genus (Silva et in Argentina (from 300 to 1200 m). It suffers al., 2004; Ferreira & Cunha, 2000; Nery from irregular reproduction linked to unseasonal et al., 2008). In vitro cultivation of seeds frosts (Zapater et al., 2009). is an appropriate method that allows the 6 N. M. Apóstolo et al., In vitro an ex vitro germination of Handroanthus propagation of species with recalcitrant dishes were previously disinfected with seed, with preservation difficulties or that sodium hypochlorite (NaClO) (55 g.L-1 have problems with asexual reproduction. active chlorine) at 50% for 5 min and rinsed After in vitro germination the seedlings with sterilized distilled water. The paper was obtained can be acclimatized directly under sterilized in an autoclave at 1 atm (120 ºC) for greenhouse conditions or used as explants in 20 min. Three repetitions were carried out for vitro morphogenesis (Blakesley et al., 1996; each species at 0, 4, 8 and 12 months since Fay, 1994; Larraburu et al., 2012; Thorpe the seed collection. The seeds were stored in et al., 1991). Therefore this technique is the fridge at 5ºC between these periods. The appropriate for ex situ conservation plans germination dishes were kept in the culture for rare or threatened species and also for chamber at 25 ± 2ºC under continuous light. massive propagation of species of economic The germination percentage was weekly interest (Silva et al., 2005; Fay, 1994). evaluated during 30 days (germination In this study the in vitro and ex vitro capacity). The germination energy was germination of three species of Handroanthus determined at five days after the beginning that occur in northwest Argentina (H. of the each test of germination. impetiginosus, H. lapacho and H. ochraceus) were evaluated. The morphology of the seed Seed culture and seedlings was also analyzed so that The seeds were stored at 5ºC for a year their characteristics could be related to the after their collection. They were disinfected germination rate in the three species. after the removal of the wing portion of the seed coat. The disinfection was done by washing with tap water for 1 h, immersion in Materials and Methods 20% NaClO for 30 min with agitation and, finally, three rinses with sterilized distilled Plant material water. Seeds of H. impetiginosus, H. lapacho and Ex vitro culture. The disinfected seed H. ochraceus (Bignoniaceae) were collected was grown in seedling trays previously from different habitats
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